ES2314088T3 - A THERMOSTABLE ISOMERASA AND ITS USE, IN PARTICULAR TO PRODUCE TAGATOSA. - Google Patents

Abstract

An L-arabinose isomerase capable of isomerizing D-galactose in D-tagatose, isomerase having at least 70% sequence identity with the sequence of SEQ ID NO: 2.

Description

A thermostable isomerase and its use, in particular to produce tagatose.

Field of the Invention

The present invention is within the field of industrial enzymes, in particular, enzymes for synthesis of sugars such as D-tagatose.

Technical background and state of the art

D-tagatose is keto sugar which corresponds to aldoazúcar D-galactose. It has a sweetener value equivalent to sucrose but of poor digestion and has been discovered to be a useful sweetener, Safe, low calorie and non-cariogenic, for products food, for which there is a great demand.

D-tagatose can be synthesized chemically, for example, as described in the US 5,002,612.

Enzymatic methods have been described for D-tagatose production. Yamanaka and Wood (1966) list a number of lactic acid bacteria that provide an enzyme L-arabinose-isomerase capable of producing ketoses from L-arabinose, D-galactose or D-fucose.

US 6,057,135 discloses a process for the manufacture of D-tagatose, where a galactose permeate is hydrolyzed to obtain a lactose hydrolyzate comprising D-galactose and glucose. The hydrolyzate is fermented to convert glucose into ethanol which is subsequently removed and the remaining D-galactose solution is subjected to enzymatic isomerization with an L-arabinose isomerase to obtain D-tagatose. The L-arabinose-isomerase preparations used are crude biomass extracts of Lactobacillus pentosus, Bacillus amyloloquefaciens or Arthrobacter spp.

WO 00/68397 describes the document using E. coli prepared for enhanced expression of L-arabinose isomerase from E. coli for the production of tagatose.

WO 02/50282 describes the isolation of a thermostable L-arabinose isomerase capable of isomerizing galactose. The amino acid sequence of this enzyme is closely related to the previously known L-arabinose-isomerase sequences, especially Bacillus stearothermophilus.

However, to date, enzymatic methods for the production of tagatose have not been used commercially. There is a high demand for new and improved low-calorie sweeteners and, consequently, improved methods for the production of tagatose with performance and efficiency are needed in the industry
superior.

At present, it has been isolated and characterized a new enzyme active in L-arabinose isomerase. This enzyme exhibits a low sequence similarity when compared to all L-arabinose-isomerase sequences known today, including those disclosed in the WO 00/68397 and in WO 02/50282. The enzyme of the current invention has a different substrate specificity in comparison with L-arabinose-isomerases from the state of technique, and it is a versatile aldose-isomerase capable of structurally isomerizing related aldoses. The enzyme can be obtained from a thermophilic microbial source and can be used, thus, at high operating temperatures.

Compendium of the invention

In a first aspect of the invention, provide a L-arabinose-isomerase, capable of isomarizar D-galactose in D-tagalose, whose isomerase has at least 70% Identity with the sequence of SEQ IDNO: 2.

A second aspect of the invention is the provision of nucleic acid encoding this L-arabinose isomerase or its active fragment of L-arabinose isomerase.

In another aspect, a construction is provided of nucleic acid comprising the above nucleic acid.

In another aspect of the invention, it is provided a cell that is transformed with the construction or with the acid nucleic mentioned above.

In another aspect, a method is provided for converting an aldose into a ketose, which comprises contacting the aldose with the isomerase of the invention and maintain the reaction under conditions where at least 1% by weight of the tile is become the corresponding ketose.

The invention provides, in another aspect, a method for the production of L-arabinose isomerase, which comprises  transform a cell with the nucleic acid of the invention and the operable link thereof to appropriate expression signals that direct isomerase expression and, optionally, sequences which direct the secretion of isomerase, propagate said cell transformed and harvest progeny cells that contain the isomerase or, if secreted into the medium, isomerase excreted.

In another aspect, the invention provides a composition comprising the isomerase of the invention in another immobilized form

Detailed description

In accordance with the foregoing, the invention described herein provides an active enzyme of L-arabinose isomerase or its active isomerase fragment, which is derived from a Thermoanaerobacter species.

The L-arabinose isomerase (EC 5.3.1.4) also known as L-arabinose-ketol-isomerase, It is in the general class of oxidoreductases intramolecular and more specifically, in the group of aldose-isomerases, which are capable of inter-convert aldosas in your ketoses corresponding. The L-arabinose isomerase is classified  and named according to its ability to convert the aldosa-L-arabinose in its corresponding ketose, L-ribulose.

The term "isolated", as used herein, refers to the material being removed from its original environment (for example, the natural environment where the material occurs naturally). For example, a polynucleotide or a polypeptide, while present in a living organism, is not isolated, but the same polynucleotide or polypeptide, which is separated from part or all of the materials coexisting in the natural system, it is isolated. Sayings polynucleotides could be part of a vector and / or said polynucleotides or polypeptides could be part of a composition, and still be isolated in the sense that the vector or the Composition is not part of the natural environment.

The genus Thermoanaerobacter includes a range of species such as T. acetoethylicus, T. brockii, T. cellulolyticus, T. ethanolicus, T. finnii, T. italicus, T. kivui, T. mathranii, T. siderophilus, T. subterraneus, T sulfurophilus, T. thermohydrosulfuricus and T. wiegelii . In a preferred embodiment, to which reference is now made, the isomerase enzyme or the active fragment thereof is obtained from the species T. mathranii , from which a useful strain (DSMZ 11426) can be obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ).

The isomerase of the invention preferably has at least one of the following characteristics: (i) an optimal activity at a temperature in the range of 40 to 95 ° C, preferably, in the range of 60 to 80 ° C, as in the range of 60 at 70 ° C, including about 65 ° C; (ii) an optimal activity at a pH in the range of 6 to 9, preferably, in the range of 7 to 9, as in the range of about pH 7 to 8; and (iii) is capable of isomerizing at least one aldopentose or at least one aldohexose. Aldopentoses are 5-carbon aldoses that include, for example, arabinose, ribose, xylose, and lyxose, while aldohexoses are six-carbon sugars, including alose, altrose, glucose, mannose, gulose, idose, galactose, and talose. More preferably, the isomerase has at least two of these characteristics and more preferably, all three characteristics together. Reaction temperatures of 60 ° C and higher, such as at least 65 ° C, 70 ° C, 75 ° C or 80 ° C, are preferred, as the risk of contamination is minimized due to the development of other microorganisms at said elevated temperatures. In addition, high temperatures allow the use of higher substrate concentrations due to a greater solubility of the substrate. The preferred isomerase of T. mathranii has an excellent temperature profile in this regard, with a maximum activity of approximately 65 ° C, and approximately or more than 70% of the maximum activity is retained in the temperature range of approximately 60 ° C to 75 ° C.

Other advantages of isomerization at a high temperature are:

(i) that the balance between aldose and ketosis changes to ketosis at higher temperatures. (For L-arabinosa-isomerase fra Lactobacillus plantarum , it has been reported that the amount of ketose at 10 ° C, 25 ° C or 53 ° C is 10%, 14% and 16% respectively (Heath, EC Horecker, BL Smyrniotis, PZ and Takagi, Y . (1958) J. Biol. Chem. 231: 1031-1037);

(ii) the specificity of the substrate towards D-galactose is higher with respect to L-arabinose at higher temperatures. U.S. Pat. No. 6,057,135 8 (Krafts Foods, Inc., Example 5) discloses L-arabinose-isomerase fra Lactobacillus pentosus with a relative specificity for L-ara: D-gal of 300: 1 at 35 ° C and 85: 1 at 60 ° C.

In comparison with the Known L-arabinose isomerases, the isomerases of the present invention appear to be relatively versatile, capable aldose-isomerases of isomerizing many structurally related aldoses. Preferably, the isomerases of the invention are capable of Isomerize at least all of the following: L-arabinose, D-galactose and D-fucose. These three aldosas have the same chiral configuration from C-1 to C-4. In particular, for the production of D-tagatose, isomerases capable of a efficient isomerization of D-galactose.

The isomerase of the invention derives from a thermophilic source, which gives it great activity at temperatures high, such as temperature ranges between mentioned above, for prolonged periods, and a Good stability against thermal denaturation. Is contemplated that L-arabinose isomerases related from other thermophilic sources, they will have characteristics similar suitable.

The activity of the enzyme with respect to L-arabinose and D-galactose can be tested in accordance with that described in Example 2 or through other applicable methods known in the state of the art. The specificity can be further defined through measurements of Comparative activity for other aldose substrates.

In one embodiment of the invention, the isomerase has the amino acid sequence of SEQ ID NO: 2 herein. The invention also encompasses variants and derivatives thereof, including variants and derivatives that have an isomerase activity and that show at least a 70% sequence identity with respect to this sequence, including at least a 75% sequence identity or at least 80%, as a sequence identity of at least 90%, and preferably, a sequence identity of at least 95% or 97%. For example, useful variants and derivatives can be obtained by isolation of microbial species such as those mentioned above and / or by genetic modification of naturally occurring isomerase organisms such as by site-directed mutagenesis, or for example, by insertion of a sequence encoding a affinity marker as a marker
His.

Alternative methods of providing variants of the invention include available methods of gene redistribution, for example, as described in Merz et al., Biochemistry (2000), 39: 880-889; J. Minshull Curr. Op. Chem. Biol. (1999) 3: 284-290; WO 95/22625 (Affymax Technologies NV); US 6,291,165 (Novo Nordisk A / S); US 6,132,970 and US 6,372,497 (Maxygen, Inc.). In summary, gene redistribution techniques involve the proportion of a plurality of genes or related nucleic acid sequences (eg, sequences encoding different L-arabinose isomerases of the present invention) that are randomly fragmented and then they are reattached by a reaction in which homologous fragments (or conserved regions of heterologous fragments) act as primers with each other. Variants obtained in this way can be tracked and selected based on different criteria. Redistribution techniques are particularly beneficial in this regard, since they allow the combination of different convenient properties of various related proteins. With respect to the isomerases of the present invention, the desired properties that can be combined by redistribution techniques and that can be traced with applicable methods of the prior art, include substrate specificity, temperature stability, optimum temperature, long term stability, effectiveness of expression in a selected host organism, etc.

The "sequence identity", as used herein, is calculated based on a reference sequence, (which in this instance is the sequence of SEQ ID NO: 2). Algorithms for sequence analysis are known in the state of the art, such as BLAST, described in Altschul et al., J. Mol. Biol. , (1990) 215: 403-10. In general, the default parameters with respect to, for example, a "rating matrix" and a "penalty for each gap" will be used for alignment.

As shown in Figure 2 and described in Example 1, the currently preferred isomerase of the invention derived from Thermoanaerobacter mathranii , has a low sequence identity (between 24% and 30%) with known L-arabinose isomerases, where all show a significantly superior identity to each other. This may explain differences in the kinetic characteristics and the specificity of the L-arabinose isomerases of the prior art.

Preferred isomerases herein invention have a molecular weight in the range of about 50-60 kDa, and more preferably, in the range of about 52-55 kDa, including approximately 53 kDa. However, the fragments Isomerase assets of the invention may have a molecular weight significantly less since they can contain only one small portion of a wild-type isomerase sequence necessary for a correct fold of the polypeptide and retention of activity.

The highly preferred L-arabinose isomerase derived from T. mathranii has a full length molecular weight of 53 kDa calculated based on the sequence of Table 1.2 herein (SEQ ID NO: 2). Many of the preferred isomerases of the invention, including the full-length isomerase derived from T. mathranii , have an active-state tetrameric quaternary structure, native to the protein.

The term "active isomerase fragment" generally refers to any fragment of an isomerase of the present invention, a fragment that is large enough to substantially retain the activity of the isomerase from which it is obtained. While the active arabinose-isomerase site has not been characterized in detail, a comparison with the related L-fucose-isomerase of E. coli (Seemann, JE and Schulz, GE (1997) J. Mol. Biol. 273: 256 -268) suggests remains of active site. Glu 337 and Asp 361 are catalytically active residues in fucose-isomerase. An alignment of known L-arabinose isomerases with L-fucose isomerase shows that the rest of glutamic acid is conserved among all known L-arabinose isomerases (Glu 300 in L-arabinose isomerase T. mathranii ). Aspartic acid is conserved in all L-arabinose-isomerase sequences except in the sequence of T. mathranii , which has a methionine in the corresponding position (Met 324) (see Figure 4). The residues that can act as proton donors are Asp 318, Glu 323 and Asp 328. Accordingly, it is postulated that the key residues of the active site are located in the region between amino acid residues 290-340. Thus, useful isomerase active fragments of the present invention may include a sequence fragment comprising residues within the region of 150-462, or for example, 200-460, such as, within the region of 200-400, or 250 -400, including the 270-350 region, or the 290-340 region. A fragment like that can be folded into a tetrameric quaternary structure, or possibly retain its activity, but with a different quaternary structure, for example, a monomeric structure. In addition, in certain embodiments, an isomerase fragment of the invention may be combined with other suitable polypeptide sequences that do not prevent isomerase activity but may improve characteristics such as, for example, on expression, solubility and / or stability, for obtaining a chimeric protein comprising an active fragment sequence of isomerase.

In useful embodiments, the isomerase of the invention has a K m value for D-galactose in the range of about 50 to 350 mM, including the range of 100 to 200 mM. Certain preferred isomerases of the invention have a similar K m also for L-arabinose. A classic definition of K_ {m} can be found in Stryer, L Biochemistry , 3rd edition, Freeman, New York, 1988. It follows that the substrate affinity of said preferred isomerases for D-galactose compared to L-arabinose, is high with respect to the prior art isomerases, as shown in Figure 2.1 of the present specification.

Preferably, the isomerase according to the invention It has a D-galactose activity that is approximately 10 to 50% of its activity of L-arabinose, as for example, in the range of between 15-30%, including the range of 20-25%

Preferred isomerases herein invention show high conversion efficiency for the D-galactose conversion in D-tagatose, even at high concentrations of substratum. Preferably, the isomerases of the invention are able to convert at least 20% by weight of D-galactose that is in a concentration of about 30% by weight or greater in the reaction medium, in A period of 24 hours.

The invention provides, in another aspect, a L-arabinose isomerase capable of isomerize D-galactose in D-tagatose, isomerase that has an identity of sequence of at least 70% with respect to the sequence of SEQ ID NO: 2 of  the present memory, or higher, as r a sequence identity at least 80% or at least 90% sequence identity with SEQ ID NO: 2 and an active isomerase fragment thereof. particular embodiments, the isomerase can have an identity of even higher sequence than SEQ ID NO: 2, as a sequence identity of at least 95% or at least 97%.

Preferably, the isomerase is derived from a thermophilic organism, such as a bacterium that is taxonomically related to the genus Thermoanaeorobacter .

In another aspect, the invention provides a nucleic acid encoding an L-arabinose isomerase or an active L-arabinose isomerase fragment thereof, selected from the group consisting of: (i) an isolated wild type nucleic acid of a Thermoanaerobacter species and (ii) a nucleic acid sequence capable of hybridizing with the sequence mentioned above under restrictive conditions.

The expression "nucleic acid", of the form used herein, includes DNA (e.g., DNA genomic or cDNA), and RNA, with natural nucleotides such as contain one or more modified bases. Thus, DNA or RNA with main structures modified for stability or by others reasons, they are nucleic acids as defined herein memory. In addition, DNAs or RNAs that comprise unusual bases, as inosine, or modified bases, as tritilated bases, only for name two examples, they are nucleic acids the way you use the expression herein.

The expression "restrictive conditions" in This context refers to the general conditions of discharge restrictivity The term "restrictivity" is known in the state of the art and is used in reference to the conditions (temperature, ionic strength and presence of other compounds such as organic solvents) under which the nucleic acid hybridization. With conditions of "high restrictivity "only acid base couplet will occur nucleic among nucleic acid fragments that have a high base complementarity frequency, compared to "weak" or "low" restrictive conditions.

As an example, the hybridization conditions of high restrictivity include (1) employing a low ionic strength and a high wash temperature, such as 0.015 M NaCl / sodium citrate 0.0015 M, pH 7.0 (0.1 x SSC) with 0.1% sodium dodecyl sulfate (SDS) at 50 ° C; (2) use 50% formamide during hybridization (v / v) with 5x Denhardt solution (0.1% by weight / volume), highly purified bovine serum albumin / 0.1% (by weight / volume)  Ficoll / 0.1% (by weight / volume) polyvinyl pyrrolidone), 50 mM phosphate buffer of sodium with a pH of 6.5 and 5xSSC at 42 ° C; or (3) use the 50% formamide hybridization, 5xSSC, 50 mM sodium phosphate  (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt solution, Sonic salmon sperm DNA (50 µg / ml), 0.1% SDS, and 10% dextran sulfate at 42 ° C with washings at 42 ° C in 0.2xSSC and 0.1% SDS.

In a preferred embodiment, the nucleic acid encoding said isomerase or fragment thereof is wild-type nucleic acid isolated from Thermoanaerobacter mathranii , or a sequence capable of hybridizing with said sequence under restrictive conditions.

In a useful embodiment, the nucleic acid of the invention encodes the amino acid of SEQ ID NO: 2 of the present memory, or an active isomerase fragment thereof. Do not However, acids are also encompassed in the present invention. nucleicides encoding active isomerase polypeptides with a high sequence identity to SEQ ID NO: 2 or a part of isomerase active of it, as with an agreement sequence identity as previously defined at least 75%, and preferably, 90% or more, such as 95% or 97%.

A particular embodiment of the invention provide the nucleic acid that has the sequence of SEQ ID: 1, shown in Table 1.1, or a fragment thereof that is encodes for an active isomerase fragment.

In another very useful aspect, the invention provides a nucleic acid construct comprising the nucleic acid of the invention. An "acid construction nucleic ", as used herein, includes a plasmid, virus, retrovirus, bacteriophage, transposon, cosmid, artificial chromosome (bacterial or yeast), which is capable of replicate in a host cell and that typically has one or more restriction endonuclease recognition sites where The sequence can be cut in a predetermined way. The construction may also contain a suitable marker to be used in the identification of transformed cells, by example, a tetracycline resistance or a resistance to ampicillin

The invention also encompasses a cell that is transformed by either the nucleic acid or construct described above. A "cell," as used herein, refers to any prokaryotic or eukaryotic cell that is used as a receptor for recombinant polynucleotides and constructs provided herein. In preferred embodiments, the transformed cell of the invention is a bacterial cell, a yeast cell or a filamentous fungus cell. A host cell, such as E. coli, can be used in this regard; however, preferred host cells are those that are easily compatible with food production. Examples of suitable bacterial host cells are: Bacillus spp, for example, Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkatophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lausus, Bacillus lausus, Bacillususus Bacillususus, Bacillususus Bacillususus, Bacillususus Bacillususus, Bacillususus, Bacillususus Bacillususus Bacillususus Streptomyces lividans or Streptomyces murinus, or Lactococcus spp., Or Lactobacillus spp. and Zymomomas spp. Useful yeast host cells include Saccharomyces spp cells. (in particular, S. cerevisiae ), Schizosaccharomyces spp., and Pichia spp., and useful cells of filamentous fungi include those of Aspergillus spp, such as A. niger, A. nidulans and A. oryzae, Mucor spp., for example, Mucor circinelloides , and Neurospora spp., for example, Neurospora crassa.

The methods to transform cells like those mentioned above with a nucleic acid or construction like those mentioned above, are fine known in the state of the art. Construction or acid nucleic can be introduced into host cells using any suitable method (e.g. electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or others substances; microprojectile bombardment, lipofection, infection, transduction).

Another aspect of the invention provides a method of converting an aldose into a ketose, method comprising  put the tile in contact with the L-arabinose isomerase of the invention and keep the reaction under conditions where convert at least 1% by weight of the tile. Preferably, the conditions are selected such that at least 10% by weight of the aldose substrate becomes its corresponding ketosis, and more preferably, that it becomes at least about 20%, including about 25%, and more preferably still, at least approximately 30% or more of the aldose in ketose.

The tile can be any of those mentioned above and preferably selected among the group consisting of L-arabinose, D-galactose and D-fucose. A very useful embodiment of the invention uses D-galactose as aldose, in which case the product of Ketose reaction is D-tagatose. The D-galactose can be easily obtained hydrolyzing lactose, obtained commercially from whey Cheese and / or milk.

In a preferred embodiment of the method, the reaction occurs at a temperature of at least 60 ° C, as in the 60-100 ° C range, including approximately 60-801 C, and preferably, in a range of approximately 60-70 ° C.

Manganese ions (Mn + 2) may be required to sustain the activity of at least some of the isomerases of the invention. Consequently, in useful embodiments of the invention, an Mn + 2 is present in the mixture of the reaction in a concentration in the range of approximately 1-2 mM, including approximately between 1-10 mM as in the range of approximately 2-5 mM. However, some of the Preferred isomerases still retain substantially all of their activity if Fe + 2 ions are present at a concentration like the one just mentioned, or higher.

Generally, high substrate concentrations are advantageous in the industrial production of D-tagatose using the method of the invention, such as 5% by weight or more, preferably 10% by weight or more and more preferably, 30% by weight. or higher, as in the range of 30-60% by weight. As discussed above, the isomerases of the present invention have a good conversion efficiency of D-galactose at said substrate concentrations, and high operating temperatures benefit a high solubility of
substratum.

The L-arabinose isomerase may be adequately provided as an isolated enzyme preparation. Can easily be selected, isolation methods of isomerase from its source, and the person skilled in the art can adjust them to obtain the enzyme isolated from the desired purity. Such methods they may comprise one or more chromatographic purification steps by ion exchange, affinity, and permeation chromatography in gel, and / or may include sonification, centrifugation and / or stages ultra filtration

Preferably, the isomerase is purified to a point where there is no other protein. In this context, the expression "essentially without any other protein "refers to a high degree of purity, where others proteins comprise less than 10% by weight of the preparation of purified isomerase, preferably, less than 5% by weight and more preferably, less than 3% by weight, as substantially 0% by weight of the preparation.

The enzyme of the invention can be used, advantageously, as a free enzyme, not immobilized, but in Other useful embodiments, isomerase enzyme preparation Isolated is immobilized. In the present context, the term "immobilized" usually refers to the link (covalent or non-covalent) of the enzyme with a solid matrix such as beads, fibers or a membrane, or the incorporation of the enzyme within a porous matrix, so that the contact between matrix and enzyme is retained during normal reaction conditions. The enzyme immobilization methods are known in the art, and include as an example, cross-linking with glutaraldehyde in the manner demonstrated in Example 3. Other methods Applicable are, for example, enzyme coupling in groups matrix hydroxyl which are activated, for example, by carbodiimide, carbonyl diimidazole, N, N = -disuccinimidyl carbonate (DSC), or bromide of cyanogen; and epoxidation coupling, for example, by 1,4-butanediol diglycidyl ether.

As shown by Example 3.3 of herein, it is observed that the immobilized isomerase of the invention has excellent long term stability during repeated reaction cycles at operating temperatures relatively high (65ºC), and thus, it is highly suitable for industrial applications

It has been discovered that a two-stage process of (i) hydrolyzing lactose into glucose and galactose and of (ii) isomerizing the galactose obtained in tagatose, can be carried out in a simple reactor; that is, that a suitable active lactase enzyme and an L-arabinose isomerase of the present invention can be used simultaneously in a reaction unit that is fed with lactose to obtain tagatose. An embodiment of this is demonstrated and described in detail in Example 4. Surprisingly, the glucose obtained does not impede the activity of the isomerase, and the glucose can be separated from the tagatose as well as any isomerized galactose through suitable separation means. , as by chromatography. A two-stage reactor procedure like this is conveniently established through the use of immobilized lactase and immobilized L-arabinose isomerase, where both enzymes can be immobilized, for example, in the manner described herein. Preferably, the lactase should maintain its activity at a high temperature, for example, in the range of 60-100 ° C, including the range of 60-80 ° C and 60-70 ° C, so that the high temperature regime of the isomerase can be used , in accordance with what is described herein. A preferred lactase enzyme in this aspect is β-glycosidase which is readily available from various sources, for example, derived from thermophilic bacteria and expressed in a suitable host such as E. coli , but more preferably, in a production host cell. of better approved and more compatible foods, such as those mentioned herein.

Based on the findings above mentioned, the description provides, in this way, in another aspect, a method for the production of D-tagatose comprising lactose hydrolyzing by lactose contact with an active lactase enzyme (in most cases, a β-galactosidase) to yield glucose and D-galactose, and convert at least a portion of the D-galactose obtained in D-tagatose by contact of galactose with an enzyme of L-arabinose-active isomerase, where said active lactase enzyme and said enzyme of L-arabinose-active isomerase is they are contained in the same low reactor unit essentially the same reaction conditions. In realizations particularly useful, the reaction conditions include a temperature in the range of about 50 ° C at about 100 ° C, preferably, about 55 ° C at about 100 ° C, and more preferably, of about 60 ° C to about 100 ° C, as in the range of approximately 60 ° C to 80 ° C, including the range of about 65 ° C to about 80 ° C, as in the range from about 65 ° C to about 75 ° C, including about 65 ° C, about 70 ° C and about 75 ° C. In addition, the reaction conditions will typically include such conditions as described above, such as a pH in the range of 6 to 9, preferably in the range of 7 to 9, as in the range of about 7 to 8.

D-tagatose produced by methods of the present invention, find use in a variety of Pharmaceutical applications, food and functional foods. Be it's a low-calorie full volume sweetener that can advantageously, completely or partially, replace sugar and / or sugar-free sweeteners in conventional sweet products such as candies, chocolate, cereals, sweet dairy products (ice cream, yogurt, milk-based drinks), baked goods, and soda. D-tagatose can also be used in bars Dietary, sugar-free chewing gum, and as a sweetener filling in medicinal products such as pills, tablets and liquid mixtures. D-tagatose is non-cariogenic and has properties Probiotics that promote healthy digestion. The compound is Safe to be used by diabetic people.

In another aspect of the invention, it is provided a method for the production of L-arabinose isomerase, which comprises transforming a cell like those described with above, with a nucleic acid of the invention, and the bond operable thereof with appropriate expression signals that direct the Isomerase expression, and optionally, sequences that direct Isomerase secretion, propagate the transformed cell of this way and harvest the progeny cells that contain the isomerase or, if secreted in the medium, the extracted isomerase. He term "transformation" refers to the change of a form inheritable from the characteristics of a host cell as response to the exogenous DNA introduced, which may or may not be integrated (covalently linked) to the chromosomal DNA that forms the genome of a cell. In prokaryotes and yeast, for example, exogenous DNA It can be maintained in an episomal element such as a plasmid. Regarding eukaryotic cells, a stably cell transformed is one where exogenous DNA has been integrated into a chromosome so that it is inherited by sister cells through of chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones formed by a population of sister cells with exogenous DNA content.

In useful embodiments, the method of invention comprises the other purification step of L-arabinose isomerase cell progeny or the means of obtaining a preparation of L-arabinose isomerase, for example, with purification methods such as those described with anteriority.

In one embodiment, the method comprises the additional step of drying the isomerase preparation at a moisture content of 10% by weight maximum. A preparation as this may be in the form of powder or granules, which may be re-dissolved in a medium to use the isomerase of the form described herein.

Still in another aspect, the invention provides a composition comprising the L-arabinose isomerase described in the present, in an immobilized form, for example, a form immobilized as described herein.

The invention is further illustrated in greater detail. in the following non-limiting examples and in the drawings, where:

- Figure 1 is a genetic map of the T. mathranii DNA fragment that was cloned into E. coli , as described in Example 1;

- Figure 2 shows the percentage of identical amino acid residues discovered by pairwise alignment of a number of known araA sequences compared to T. mathranii sequences. Known sequences are identified by access numbers of the EMBL / Genbank database;

- Figure 3 shows a phylogenetic tree based on all the L-arabinose-isomerase amino acid sequences currently available in public sequence databases and the sequence of T. mathranii ;

- Figure 4 shows an amino acid sequence alignment of the AraA gene of Thermoanaerobacter mathranii and other araA sequences currently available in public databases. The numbers shown below the sequences refer to the araA sequence of T. mathranii . The catalytically active amino acid residues at the active site of L-fucose-isomerase E. coli are Glu 337 and Asp 361, and the corresponding putative amino acids are marked with "#" in the alignment of the L-arabinose-isomerase sequences . The conserved amino acid residues are marked with "*", the conservative substitutions are marked with ":" and the related residues are marked with ".". The alignment was done with the CLUSTAL X program (1.8);

- Figure 5 illustrates the temperature dependence of T. mathranii L-arabinose isomerase produced in E. coli ;

- Figure 6 illustrates the pH dependence of T. mathranii L-arabinose isomerase produced in E. coli ; Y

- Figure 7 illustrates the reactor conversion lactose in tagatose with immobilized lactase and isomerase immobilized

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Example 1 Cloning of the L-arabinose-isomerase ( araA ) gene from Thermoanaerobacter mathranii and heterologous enzyme production in E. coli

Thermoanaerobacter mathranii DSMZ 11426 thermophilic anaerobic microorganism was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascherodes Weg 1b, D-38124 Braunschweig, Germany (DSMZ). Originally, the strain was described as "strain A3" (Sonne-Hansen, J. Mathrani, IM and Ahring, BK (1993) Appl. Microbiol. Biotechnol. 38: 537-541). Subsequently, the strain was deposited in DSMZ and the name T. mathranii (Larsen, L., Nielsen, P. and Ahring, B. (1997) Arch. Microbiol. 168: 114-119) was proposed.

1.1 Growth of T. mathranii

T. mathranii (DSMZ 11426) was grown at 65 ° C under anaerobic conditions in the medium recommended by DSMZ. After growth, the culture was centrifuged and the pellet was stored at -80 until purification of chromosomal DNA.

1.2 Gene Cloning

A classical phenol / chloroform extraction method described by Sambrook et al. Was used for the purification of total chromosomal DNA from frozen T. mathranii cells. Purified chromosomal DNA was partially cleaved with restriction enzyme Sau3A (New England Biolabs) and approximately 3-4 kb DNA fragments were purified from agarose gels using the GFX Gel Band Purification Kit (Amersham Pharmacia Biotech) .

The plasmid of pBluescript KS (+/-) (Strategene Cloning Systems) was restrained with restriction enzyme Bam HI (New England Biolabs), treated with alkaline phosphatase (CIP, New England Biolabs) and purified from agarose gels using the GFX Gel Band Purification kit (Amersham Pharmacia Biotech). After ligation of purified DNA fragments and the purified plasmid vector, the ligation mixture was introduced into supercompetent DH10B cells (Life Technologies) by electroporation as described by the manufacturer. The transformed cells were plated on LB medium containing ampicillin (100 µg / ml). Approximately 16,000 colonies of 20 plates were pooled, and a Jetstar kit (Genomed) was used to produce a plasmid preparation from the pooled cells.

A plasmid bank prepared from cells grouped by electroporation was introduced into UP1089 cells, an Escherichia coli strain carrying an araA mutation that prevents it from developing in a minimal medium of L-arabinose. Strain UP1089 was provided by E. coli Genetic Stock Center, 355 Osborn Memorial Laboratories, Department of Biology, PO Box 208104, Yale University, New Haven, CT 06520-8104, USA. After transformation with the plasmid bank, the UP1089 cells were plated on minimal medium with L-arabinose content as the only sugar, and thus selected to complement the araA mutation. Approximately 90 colonies were obtained by development selection on minimal medium plates of L-arabinose.

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1.3 DNA sequence and phylogenetic comparison

Plasmid inserts were selected from two of these colonies to sequence DNA (ALF Express, Amersham Pharmacia Biotech), and were found to contain identical DNA fragments that comprised an L-arabinose-isomerase ( araA ) gene followed by a gene of L-ribulokinase ( araB ) (Figure 1). The nucleotide sequence of the 5 'part of a cloned DNA fragment comprising the araA and araB genes is shown in Table 1.1 below. The open reading frame of the araA gene is shown in bold (nucleotides 463-1860).

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TABLE 1.1 Nucleotide sequence of the DNA fragment comprising the araA and araB genes of Thermoanaerobacter mathranii (SEQ ID NO: 1)

one

2

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The open reading frame of the araA gene encoded 465 amino acid residues, corresponding to a molecular weight of 52,785 Da. The amino acid sequence of the araA gene product (SEQ ID NO: 2) deduced from the sequence of Table 1.1 is shown in Table 1.2 below.

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TABLE 1.2 Amino acid sequence of T. mathranii DSMZ 11426 araA gene product (SEQ ID NO: 2)

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3

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The amino acid sequence showed a homology with the previously known L-arabinose isomerases. The percentage of identical amino acid residues was relatively low, 24% and 30%, when the sequence of T. mathranii aligned with other araA genes (Figure 4). In comparison, the level of identity was above 46% within the reference group of previously known L-arabinose isomerases (Figure 2). The L-arabinose-isomerase amino acid sequences currently available in public sequence databases and the T. mathranii sequence were subsequently used for the construction of a phylogenetic tree (Figure 3) using the ClustalX program (Thompson, JD , Gibson, TJ, Plewniak, F., Jeanmougin, F. and Higgins, DG (1997) Nucleic Acids Res . 25: 4876-4882).

One of the two colonies was selected for a further use

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1.4 Heterologous production of L-arabinose-isomerase from T. mathranii in E. coli cells UP1089

E. coli cells that harbor the L-arabinose-isomerase gene were grown overnight at 37 ° C in LB medium containing ampicillin (100 µg / ml). After centrifugation, the cells were resuspended in 50 mM Tris-Cl, with a pH of 7.5 and lysed in a French pressure cell operated at 1,100 psig. The cell debris was removed by centrifugation and the resulting cell extract was used to characterize the enzyme, as described below.

Example 2 Characterization of T. mathranii L-arabinose isomerase produced in E. coli 2.1 Test method

The activity of L-arabinose isomerase was determined according to that described by Yamanaka, K. and Wood, WA (1996) in Methods in Enzymology 9: 596-602. An enzyme sample, 10 and 50 µl, was mixed with 950 µl of test reagent and incubated at 65 ° C for 60 minutes. The final concentrations were: L-arabinose, 5 mM; MnCl2, 5 mM; maleate buffer, with a pH of 6.9, 25 mM. When galactose or fucose was used as a substrate, the concentration of D-galactose or D-fucose was 0.5 M and the incubation time was approximately 16 hours.

The concentrations obtained from the ketoses L-ribulose, D-tagatose or D-fuculose, respectively, were determined by the method of cysteine-carbazol-sulfuric acid (Dische, Z. and Borenfreund, E. (1951) J. Biol. Chem . 192: 583-587). Samples were incubated at room temperature for 60 minutes and the absorbance at 560 nm was measured in a microplate reader. Standard curves were performed showing the color response of 0 to 5 mM ketose with D-tagatose and D-ribulose, since L-ribulose was not commercially available. (No standard curve was obtained for D-fuculose that was not commercially available.)

Lactose concentrations were determined, D-glucose, D-galactose, D-tagatose, L-ribulose and D-fuculose by high pressure liquid chromatography using an Aminez HPX-87C column (Bio-Rad) and a refractive index detector. Be de-ionized the mobile phase, water was degassed, the column temperature was 85 ° C and the flow rate was 0.6 ml / min.

2.2 Temperature dependence

D-galactose assays that were carried out at increasing temperatures between 45 ° C and 85 ° C, showed the highest activity at 65 ° C (Figure 5).

2.3 pH dependence

D-galactose assays that were carried out at a pH of 4, 5, 6, 7, 8 and 9, they showed, respectively, the highest activity at a pH of 8 (Figure 6). He buffer used for these tests was a mixture of acetic acid 25 mM, 2- [N-morpholino] ethanesulfonic acid (MONTH) 25 mM, 5 mM MnCl2, and 25 mM tris [hydroxymethyl] -aminomethane (TRIS), which was titrated with HCl or NaOH.

2.4 Metal ion requirement

A range of metal salts was tested (MnCl2, NaCl, KCl, MgSO4, ZnSO4, CuSO4 and FeSO_4) for its ability to reactivate enzymes that had been previously dialyzed against a buffer solution that It contained 5 mM ethylenediaminetetraacetic acid (EDTA). Only the adding MnCl2 (100%) or FeSO4 (76%) restored the activity of L-arabinose isomerase.

Other Escherichia coli L-arabinose-isomerase enzymes have also been reported (Patrick, JW, and Lee, N. (1968) J. Biol. Chem. 243: 4312-4318), Aerobacter aerogenes (Yanamaka, K. and Wood, WA (1966) Methods in Enzymology 9: 596-602), and Lactobacillus gayonii (Nakamatu, T. and Yamanaka, K. (1968) Biochim. Biophys. Act 178: 156-165) require Mn 2 +}.

2.5 Molecular Weight

A native molecular weight of approximately 220 kDa by gel filtration on a column Superdex 200 HR10 / 30 (Amersham Pharmacia Biotech) and by test of Enzyme of collected fractions.

Sodium dodecylsulfatopolyacrylamide gel (SDS-PAGE) electrophoresis of collected fractions showed a subunit molecular weight of approximately 55 kDa, which corresponds to the expected 53 kDa subunit size of the DNA sequence of the open reading frame of araA (465 amino acid residues). The identity of the 55 kDa L-arabinose-isomerase band seen in SDS-PAGE was verified by electrophoretic transfer of the polyvinylidene difluoride membrane polypeptide (PVDF) and the N-terminal amino acid sequence in an automated micro-sequencer. The N-terminal sequence, MQTKKK-, was identical to the amino acid sequence deduced from the DNA sequence of the T. mathranii araA gene, as described previously in Example 1.

The observed native molecular weight of approximately 220 kDa and the subunit size of approximately 55 kDa suggest that the active enzyme is a tetramer. The corresponding enzyme in E. coli is a hexamer containing six identical subunits of approximately 60 kDa (Patrick, JW and Lee, NJ (1969) J. Biol. Chem. 244: 4277-4283).

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2.6 Substrate specificity

It has been previously reported that certain L-arabinose-isomerase enzymes can isomerize not only L-arabinose, but also D-galactose and D-fucose (Yamanaka, K. and Wood, WA (1966) Methods in Enzymology 9: 596-602 ). These sugars have the same configuration in C 1 to C 4, including an L-cis to C 2 -C 3 configuration.

The molar ratio of keto sugar generated by isomerization of L-arabinose and D-galactose respectively, was determined at a substrate concentration of 0.5 M (with a pH of 6.9 at 65 ° C). Be calculated the relationship from color responses of cysteine-carbazol-sulfuric acid and curves standard generated with ribulose and tagatose, respectively. This calculation showed that D-galactose activity was 21% of L-arabinose activity.

The color response obtained with 0.5 M of D-fucose was 46% of the corresponding response view with L-arabinose. The molar relationship between D-fucose and L-arabinose activity could not be calculated, since no standard curve was obtained for D-fuculosa.

No enzyme activity was detected. significant with other aldohexoses (D-glucose, D-mannose), with other aldopentoses (D-arabinose, L-ribose, D-ribose, D-xylose), nor with another deoxiazúcar (L-fucose).

2.7 Substrate affinity

The apparent Michaelis-Menten constants, K m , of the enzyme were approximately 80 mM for L-arabinose, approximately 120 mM for D-galactose, and approximately 145 mM for D-fucose. Table 2.1 shows previously recorded values for other L-arabinose isomerases. The broad substrate specificity shown above and similar K m values for L-arabinose, D-galactose, and D-fucose suggest that the enzyme of the invention, compared to other L-arabinose isomerases, is an aldose - Versatile isomerase capable of isomerizing a range of structurally related aldoses.

TABLE 2.1 Substrate affinity of L-arabinose isomerases: K m values for L-arabinose, D-galactose and D-fucose

4

References cited in Table 2.1:

1: Yamanaka , K. and Wood , WA ( 1966 ) Methods in Enrymology 9: 596-602.

2: Ibrahim , OO and Spradlin , JE, U.S. Patent No. 6,057,135 (Kraft Foods, Inc.).

3: Patrick , JW and Lee , N. ( 1968 ) J. Biol. Chem. 243: 4312-4318.

4: Heath , EC, Horecker , BL, Smymiotis , PZ and Takagi , Y. ( 1958 ) J. Biol. Chem. 231: 1031-1037.

5: Nakamatu , T. and Yamanaka , K. ( 1969 ) Biochim. Biophys Minutes 178: 156-165.

6: Izumori, K., Yeda, Y. and Yamanaka, K. (l978) J. Bacteriol 133: 413-414.

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2.8 Enzymatic bioconversion of D-galactose in D-tagatose with enzyme not immobilized

Free enzyme was used to demonstrate the bioconversion potential of the enzyme at high substrate concentrations. One ml test mixtures containing 0.20 ml of E. coli cell extract were incubated with T. mathranii recombinant L-arabinose isomerase, 0.30 g of D-galactose (30%, 1.67 M) or 0.60 g of D-galactose (60%, 3.33 M), 25 mM maleate buffer, with a pH of 6.9 and 5 mM of MnCl2. Control samples without enzyme were treated similarly. Periodically, samples were taken and the concentration of D-tagatose was determined by the cysteine-carbazole-sulfuric acid method as described previously. The results appear in Table 2.2 A (30% D-tagatose) and in Table 2.2 B (60% D-galactose).

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TABLE 2.2 A

Bioconversion of D-galactose in D-tagatose with enzyme free  Initial concentration of D-galactose 30% (1.67 M)

5

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TABLE 2.2 B

Bioconversion of D-galactose in D-tagatose with enzyme free  Initial concentration of D-galactose 60% (3.33 M)

6

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Example 3 D-galactose bioconversion in D-tagatose with immobilized enzyme 3.1 Enzyme immobilization by crosslinking with glutaraldehyde and polyethyleneimine

Cells from a 2 liter culture of E. coli cells producing L-arabinose isomerase were collected by centrifugation and homogenized in a French pressure cell as described above. The enzyme was immobilized by cross-linking all cellular components with glutaraldehyde and polyethyleneimine in accordance with that described in US 4,355,105. Glutaraldehyde, 25% (weight / volume) was obtained from Merck, Darmstadt, Germany, and 50% polyethyleneimine (weight / volume) was obtained from Sigma Chemicals. The crosslinked enzyme was coated by centrifugation, and the pellet was lyophilized and stored at 4 ° C until later use.

The activity of the immobilized enzyme was determined by incubation of 20 mg of lyophilized enzyme in a one ml test mixture containing 0.30 g of D-galactose (30%, 1.67 M), 25 mM maleate buffer, pH 6.9, and 5 mM of MnCl 2 at 65 ° C. A control sample without enzyme was treated similarly. Periodically, samples were taken and the concentration of D-tagatose was determined by high pressure liquid chromatography. The yield of generally immobilized enzyme was 60-100 units per liter of E. coli cell culture , and the recovery of enzyme activity after immobilization was approximately 50%. The specific activity of the immobilized enzyme preparation was approximately 55 units per gram of lyophilized enzyme. One unit was defined as the amount of enzyme that produces a micromol of D-tagatose per minute at 65 ° C, pH 6.9, in a 30% solution (weight / volume) of D-galactose.

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3.2 Enzymatic bioconversion of D-galactose in D tagatose with immobilized enzyme crisscrossed

One ml test mixtures were incubated which contain 40 mg of lyophilized enzyme (2.2 units), 0.30 g of D-galactose (30%, 1.67 M), 25 mM maleate buffer, pH 6.9 and 5 mM of MnCl 2 at 65 ° C. A control sample of enzyme similarly. Periodically, samples were taken and determined the concentration of D-tagatose by high pressure liquid chromatography. Table 3.1 shows the results obtained.

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TABLE 3.1 D-galactose bioconversion in D-tagatose with immobilized enzyme crisscrossed

7

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3.3 Long-term stability of the enzyme immobilized

A lyophilized enzyme sample (40 mg, 2.2 units) was incubated at 65 ° C in a one ml test mixture containing 0.30 g of D-galactose (30%, 1.67 M), 25 mM buffer maleate, pH 6.9 and 5 mM of MnCl 2. Samples were taken to determine the concentration of D-galactose at 0 h and 24 h, and analyzed by HPLC as described above. After sampling at 24 h, the sugar solution on the immobilized enzyme was removed, and a new test solution was added to a final volume of 1.0 ml. The 24 h incubation was repeated four times using the same enzyme sample for all galactose conversion cycles in
Tagatose

The final concentration of tagatose after Conversion for 24 hours remained constant (average of 529 mM ± 5%) during repeated bioconversions with the same sample of enzyme (Table 3.2), and even after the operation during more than 100 h at 65 ° C, the enzyme showed no sign of speed reduced reaction. The experiment showed that the enzyme immobilized is a stable biocatalyst that can be used for multiple repeated bioconversions of galactose in tagatosa.

TABLE 3.2 Long-term stability of the enzyme immobilized

8

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Example 4 Conversion of simple lactose reactor into tagatose with lactase immobilized and immobilized isomerase

The combined use of a thermostable immobilized lactase and a thermostable immobilized L-arabinose isomerase was demonstrated for direct high temperature conversion of lactose into tagatose in a single reactor. The enzyme selected for lactose hydrolysis was the extremely thermostable β-glycosidase of Sulfolobus solfataricus of thermoacidophilic archae (Moracci, M., Ciaramella, M. and Rossi, M. [2001] Methods in Enzymology 330: 210-15). This broad-spectrum enzyme is an efficient lactase, and has been successfully cloned and expressed in E. coli and other host microbial organisms ( ibid .).

While most of the previously characterized lactose are strongly inhibited by galactose, it has been reported that this particular enzyme is not inhibited by galactose, and is only moderately inhibited by glucose (Pisani, FM, Rella, R., Raia, CA, Rozzo, C., Nucci, R., Gambacorta, A., De Rosa, M. and Rossi, M. [1990] Eur. J. Biochem. 187: 321-328).

Pisani et al 1990). It was expected that these favorable properties with respect to the inhibition of a final product would be highly advantageous for the hydrolysis of lactose at a high substrate concentration such as 30% (weight / volume). In addition, the S. solfataricus enzyme does not require any cofactor, unlike, for example, E. coli? -Galactosidase which is Mg2 + dependent. Therefore, only the cofactor required by the T. mathranii isomerase manganese ions has to be included in the reaction medium, thus excluding any influence of a cofactor required by lactase.

4.1 Heterologous production in E. coli and immobilization of β-glycosidase from Sulfolobus solfataricus

The gene encoding Sulfolobus sulfataricus β-glycosidase was cloned and expressed in E. coli . The gene was isolated by polymerase chain reaction (PCR) using purified chromosomal DNA from strain P2 of Sulfolobus solfataricus . Primers were designed containing additional restriction sites for Nd el and BamH I to give rise to the entire coding sequence in a fragment that was subsequently cloned, in the standard expression plasmid pET3a (Novagen).

E. coli cells that produce the enzyme were cultured, collected by centrifugation, lysed in a French pressure cell and crosslinked with glutaraldehyde and polyethyleneimine as described in US 4,354,105. The immobilized enzyme was recovered by centrifugation and lyophilization of the pellet. The activity of immobilized lactase was 1,500 units / g in dry weight. One unit was defined as the amount of enzyme that releases one micromol of glucose per minute at 65 ° C, pH 6.5, in 30% (weight / volume) of lactose solution.

4.2 Heterologous production in E. coli and immobilization of L-arabinose isomerase from Thermoanaerobacter mathranii

Thermoanaerobacter mathranii L-arabinose isomerase was produced in E. coli and immobilized as described in Example 3.1.

4.3 Conversion of a simple lactose reactor into tagatose with immobilized lactase from S. solfataricus and immobilized L-arabinose-isomerase from T. mathranii

One ml test mixtures were incubated with 20 mg (30 units) content of immobilized lactase, 80 mg (4.4 units) of immobilized isomerase, 0.30 g of lactose (30%, 875 mM), 25 mM K-maleate buffer, pH 6.9 and 5 mM of MnCl2 at 65 ° C. A control sample without enzymes from similar form. Periodically, samples were taken and determined the concentrations of glucose, galactose and tagatose by high pressure liquid chromatography. According to the Figure 7, glucose concentration increased to approximately 800 mM for 24 hours, indicating that almost all of the hydrolyzed lactose in galactose and glucose. Tagatose concentration increased linearly to approximately 300 mM for 24 hours, indicating a bioconversion of approximately 38% (300 mM / 800 mM).

Successful hydrolysis of lactose and subsequent isomerization of galactose in tagatose showed that the two enzymes involved were able to operate under them reaction conditions with respect to pH, temperature, Buffer components and metal ion concentration (5 mM of MnCl2). In addition, it was shown that the enzyme isomerase is not affected by the high concentration of glucose present as Lactose hydrolysis result.

References

- Altschul , SF, Gish , W., Miller , W., Myers , EW and Lipman , DJ ( 1990 ) J. Mol. Biol. 215: 403-10.

- Dische , Z. and Borernfreund , E. ( 1951 ) J. 8iol Chem. 192: 583-587.

- Heath , EC, Horecker , BL, Smyrniotis , PZ and Takagi , Y. ( 1958 ) J. Biol. Chem. 231: 1031-1037.

- Izumori, K., Yeda, Y. and Yamanaka, K. (1978) J. Bacteriol. 133: 413-414.

- Larsen , L., Nielsen , P. and Ahring , B. ( 1997 ) Arch. Microbiol. 168: 114-119.

- Nakamatu , T. and Yamanaka , K. ( 1969 ) Biochim. Biophys Minutes 178: 156-165.

- Patrick , JW and Lee , N. ( 1968 ) J. Biol. Chem. 243: 4312-4318.

- Patrick , JW and Lee , NJ ( 1969 ) J. Biol. Chem. 244: 4277-4283.

- Pisani , FM, Rella , R., Raia , CA, Rozzo , C., Nucci , R., Gambacorta , A., De Rosa , M. and Rossi , M. ( 1990 ) Eur J. Biochem. 187: 321-328.

- Moracci , M., Ciaramella , M. and Rossi , M. ( 2001 ) Methods in Enzymology 330: 201-15.

- Sambrook , J., Fritsch , EF and Maniatis , T. Molecular cloning: A Laboratory Manual , 2nd Edition ( 1989 ) Cold Spring Harbor Laboratory, Cold Spring Harbor , NY, USA, Seemann , JE and Schulz , GE ( 1997 ) J. Mol. Biol. 273: 256-68.

- Sonne-Hansen , J., Mathrani , IM and Ahring , BK ( 1993 ) Appl. Microbiol Biotechnol 38: 537-541.

- Thompson , JD, Gibson , TJ, Plewniak , F., Jeanmougin , F. and Higgins , DG ( 1997 ) Nucleic Acids Res. 25: 4876-4882

- Yamanaka , K. and Wood , WA ( 1966 ) Methods in Enzymology 9: 596-602.

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<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> T. maritima

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 11

         \ vskip1.000000 \ baselineskip
      

29

30

         \ vskip0.400000 \ baselineskip
      

<210> 12

         \ vskip0.400000 \ baselineskip
      

<211> 496

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> T. neapol

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 12

31

32

         \ vskip0.400000 \ baselineskip
      

<210> 13

         \ vskip0.400000 \ baselineskip
      

<211> 501

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> M. smegmatis

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 13

33

3. 4

         \ vskip0.400000 \ baselineskip
      

<210> 14

         \ vskip0.400000 \ baselineskip
      

<211> 407

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> T. mathranii

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 14

35

36

Claims (23)

1. One L-arabinose isomerase capable of isomerize D-galactose in D-tagatose, isomerase that has at least one 70% sequence identity with the sequence of SEQ ID NO: 2. 2. The isomerase of claim 1, isomerase that has at least a 90% sequence identity with the sequence of SEQ ID NO: 2. 3. The isomerase of claim 2, which It has the amino acid sequence of SEQ ID NO: 2. 4. An active isomerase fragment of the L-arabinose isomerase of any of claims 1-3. 5. A nucleic acid encoding the L-arabinose isomerase of any of claims 1-3 or the active isomerase fragment of claim 4. 6. A nucleic acid according to claim 5, selected from the group consisting of: (i) a wild-type nucleic acid isolated from a Thermoanaerobacter species and (ii) a nucleic acid sequence capable of hybridizing with the sequence of (i) under restrictive conditions, said conditions comprising washing with 0.1 x SSC with 0.1% SDS at 50 ° C. 7. The nucleic acid of claim 6, wherein the wild-type nucleic acid is isolated from Thermoanaerobacter mathranii . 8. The nucleic acid of claim 6, having the nucleic acid sequence of SEQ ID NO: 1, or a fragment thereof encoding an active fragment of isomerase 9. A nucleic acid construct that comprises the nucleic acid of claim 5 or 6. 10. The construction of claim 9, which it is a construction selected from the group consisting of a plasmid, a chromosome, a bacteriophage, a transposon and a cosmid 11. A cell that is transformed with acid nucleic of claim 5 or 6, or with the construction of the claim 9. 12. A method to convert D-galactose in D-tagatose, which comprises contacting D-galactose with the isomerase of any of claims 1-4 and keep the reaction under conditions in which it converts to minus 1% by weight of D-galactose. 13. The method of claim 12, in the that the reaction is carried out at a temperature of at least 60 ° C 14. The method of claim 12, in the that at least 10% by weight of the D-galactose substrate It becomes D-tagatose. 15. The method of claim 14, in the that at least 25% by weight of the D-galactose substrate It becomes D-tagatose. 16. The method of claim 12, wherein isomerase is provided as an enzyme preparation isolated. 17. The method of claim 16, wherein The isolated enzyme preparation is immobilized. 18. A method of producing L-arabinose-isomerase, which comprises transforming a cell with the nucleic acid of claim 5 or 6 and operably linking thereto, appropriate expression signals that direct the expression of the isomerase, and optionally, sequences that direct the secretion of the isomerase, propagate said transformed cell and collect the progeny cells containing the isomerase or, if secreted into the medium, the isomerase
excreted 19. The method of claim 18, in the that the cell that is transformed is a cell selected from group consisting of a bacterial cell, a yeast cell and a filamentous fungus cell. 20. The method of claim 18, which comprises the additional step of purifying the L-arabinose isomerase from progeny cells or the medium to obtain a preparation of L-arabinose isomerase. 21. The method of claim 20, wherein the isomerase is purified to a point where it is found essentially without any other protein.

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22. The method of claim 21, which it comprises the additional step of drying the preparation until a moisture content of 10% by weight, maximum. 23. A composition comprising isomerase of claim 1 in immobilized form.